Tips from the journals of the American Society for Microbiology

A new vaccine strategy using nanoparticles as carriers may be the key to developing a vaccine against norovirus, one of the most common causes of foodborne disease in the United States. Researchers from the Cincinnati Children's Hospital Medical Center report promising findings in the January 2011 isse of the Journal of Virology.

The application of nanoparticles as carriers to present small peptide antigens is a growing field within vaccine development. Researchers led by Xi Jason Jiang of Cincinnati Children's Hospital Medical Center, have described a new nanocarrier, called a P particle, which holds promise as a scaffold for a variety of vaccines. In the current study they inserted rotavirus antigen into the P particle, which boosted immune response to rotavirus, as well as norovirus, in mice.

Both rotavirus and norovirus are important causes of acute gastroenteritis. The former causes severe diarrhea in children, and kills an estimated 527,000 worldwide, annually. Norovirus is a notably highly transmissible, and particularly unpleasant flu, which can result in one to three days of vomiting and diarrhea in otherwise healthy adults, and which kills 200,000 children annually.

The P particle's unique feature is the scaffold. The P particle consists of 24 copies of an outer coat protein from norovirus. The beauty of the P particle is that it contains three types of surface loops, which are ideal for presenting a wide variety of antigens. Additionally, it is highly immunogenic and extremely stable, the latter an important quality for use in developing nations. The antigens can easily be inserted during the manufacturing process. Production is a simple matter of expressing the cloned P particle in E. coli.

In addition to the rotavirus antigen, the team has succeeded in inserting a number of antigens into the P particle, varying in size up to more than 200 amino acids. The resulting vaccines have induced significantly stronger immune responses in mice than have free antigens.

Jiang is principal investigator for a five year, $4.1 million grant from the National Institute of Allergy & Infectious Disease (NIAID) that Cincinnati Children's received last May to develop the P particle vaccine against norovirus. "With the unique features of high efficiency, easy production, and low cost, this new platform will find a broad application in the biomedical sciences," says Jiang.

The combination the antibiotic ceftazidime plus the compound NXL104 is active against bacterial pathogens containing genes that confer resistance to multiple carbapenems, according to two papers published in the January 2011 issue of the journal Antimicrobial Agents and Chemotherapy.

Carbapenems are the most powerful penicillin-related antibiotics, often used against difficult bacterial infections that have become resistant to other drugs. The spread of bacteria with carbapenem resistance--now throughout the world--is a grave concern, says David Livermore of the Health Protection Agency, London, UK, the principal investigator for one of the papers. There are a variety of genes encoding very different carbapenem-destroying enzymes. Two of the most important of these are KPC--which is now widespread in the US--and NDM, which is rapidly spreading internationally from India and Pakistan.

Livermore showed that NLX104 plus ceftazidime is effective in vitro against Klebsiella pneumoniae carrying the resistance gene that codes for carbapenemase, but not those with NDM; but that the antibiotic combination NXL104 plus aztreonam is active against all carbapenemase producers, including those with NDM.

"What was most impressive was the ability of the inhibitor combination [NXL104 plus ceftazidime] to treat bacteria that produced three or four beta-lactamases," says Karen Bush of Indiana University, Bloomington, who was not involved in the research.

In the other paper, Andrea Endiamiani and Robert Bonomo of the Louse Stokes Cleveland VA Medical Center and Case Western Reserve University show in animal models that ceftazidime plus NXL104 is active against Klebsiella pneumoniae carrying carbapenemase, and expressing high levels of resistance to imipenem and ceftazidime. Additionally, adding NXL104 to ceftazidime significantly increased survival of mice with otherwise lethal infection.

"To our knowledge, these are the first studies that show that NXL104 is effective when combined with ceftazidime in animal models," says Bonomo of his research. "This opens the door for more in-depth investigations into this, as well as novel derivatives."

These new antibiotic combinations are critical because the resistance they fight is spreading worldwide. The NDM resistance genes are believed to have originated in India, but they have been found in the United States, in much of Europe, Israel, Hong Kong, Japan, and Kenya, and victims include medical tourists to India and Pakistan, says Livermore. The major carriers of NDM-1, one of the resistance genes, are Klebsiella pneumoniae and E. coli, but Acinetobacter species are additional hosts, which Livermore says reflects the ease with which plasmids carrying the resistance genes can spread among different bacterial species.

Livermore warns that the combination in India of highly developed medical facilities serving both locals and medical tourists, largely unregulated use of antibiotics, a huge population, and a "creaky infrastructure" that allows circulation of gut bacteria between the sewers and the drinking water creates a "frightening" potential for local spread and international dissemination.

Transmission of prion brain diseases such as bovine spongiform enecephalopathy (BSE) - also known as mad cow disease - and human variant Creutzfeldt-Jakob disease (vCJD) is generally attributed to the consumption of the brain or organ meat of infected animals but new research demonstrates lambs exposed to milk from prion-infected sheep with inflamed mammary glands can develop prion disease as well. The research, which is published in the January 2011 issue of the Journal of Virology, has major implications for human and livestock health.

"Prions cause devastating, ultimately fatal infections in humans," says corresponding author Christina Sigurdson of the University of California, San Diego School of Medicine. "This study is the first demonstration of prions from an inflamed organ being secreted, and causing clinical symptoms in a natural host for prion disease."

Recent research had suggested that human-to-human transmission of prions has occurred via blood transfusions, "underscoring the importance of understanding possible transmission routes," the researchers write. The misfolded prions that cause vCJD in humans, and BSE in cattle -- which can be transmitted to humans -- commonly accumulate in lymphoid tissues before invading the central nervous system, where they wreak their deadly effects. Inflammation can cause lymphoid follicles to form in other organs, such as liver and kidney, which leads prions to invade organs that normally do not harbor infection. In recent research, this team, led by Ciriaco Ligios of the Istituto Zooprofilattico Sperimentale in Sardinia, Italy and Adriano Agguzi at the University of Zurich, Switzerland, reported sheep with misfolded prions in inflamed mammary glands, also known as mastitis, raising concerns that prions could be secreted into milk.

In the new research, the team infected sheep with a common retrovirus that causes mastitis, and misfolded prions. They bred the sheep, in order to stimulate the females to produce milk, which they then collected and fed to lambs that had never been exposed to prions. The lambs developed prion disease after only two years, a speed which surprised the researchers, and "suggested that there was a high level of prion infectivity in milk," says Sigurdson.

The research raises several disturbing possibilities.

A common virus in a sheep with prion disease can lead to prion contamination of the milk pool and may lead to prion infection of other animals.

The same virus in a prion-infected sheep could efficiently propagate prion infection within a flock, through transmission of prions to the lambs, via milk. This might be particularly likely on factory farms, where mastitis may be common, and could occur in goats as well as sheep.

However, "This work cannot be directly extrapolated to cattle," says Sigurdson. She says that BSE prions do not accumulate to detectible levels in lymphoid organs, and thus would not be expected to accumulate with inflammation. "Nonetheless," she says, "it would be worth testing milk from cattle with mastitis for prions as there may be other cellular sources for prions entry into milk."

A new experimental vaccine against Staphylococcus aureus has been shown to be well-tolerated, and to boost antibodies, according to a paper in the December, 2010 issue of the journal Clinical and Vaccine Immunology. The vaccine was developed by Merck.

In the study, investigators led by Clayton Harro of the Johns Hopkins Bloomberg School of Public Health, Baltimore, gave a single vaccination in one of three different doses, or placebo, to four groups of 31 healthy volunteers, each, who ranged in age from 18-55. All three doses stimulated a rise in antibodies, the two higher doses significantly more so than the lowest dose. Antibody levels reached high levels after about 14 days, and they remained at those levels after three months.

"Based on this and other studies, the vaccine is now being tested in people who are at high risk of getting infected by S. aureus to see if the resulting antibodies can protect them from disease," says Harro. The need for such a vaccine is critical. S. aureus is the leading cause of hospital-acquired infections. "Invasive S. aureus infections (blood stream, deep wound, prosthetic device) have high associated morbidity and mortality," says Harro--in the US and Europe, 6 million people become infected annually, and 140,000 die. Multidrug-resistant S. aureus is an increasing problem.

Vaccine design has been a big challenge, says Harro. "S. aureus has a complicated structure, a vast array of strains, and an uncanny capacity to evade immune surveillance systems in our bodies." But these complexities may have been rendered largely moot when the researchers discovered a single protein on the bacterial surface that is common to most S. aureus strains. Modern antigen discovery techniques, not available until recently, enabled the protein's discovery.

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